Microfluidics technology, i.e. nanoliter to picoliter size droplets on or in disposable chips for biochemical analysis, is becoming increasingly prevalent as networks of small channels have been proven to be a flexible platform for the precision manipulation of such small amounts of fluids.
One way of handling microfluidic reagents is by producing aqueous droplets in an immiscible, inert carrier fluid as disclosed in WO 2007081385, WO 2007081386, WO 2007081387, WO 2007133710 and WO 2008063227 (Raindance Technologies Inc). These suspended droplets provide a well defined, encapsulated microenvironment that eliminates cross contamination and allows for sequential cycling of reagents. They can be used to isolate reactive materials, cells, proteins, or small particles for further manipulation and study, and droplets containing specific properties can be selectively removed from the droplet population and collected.
For microbiological applications using cellular expression of proteins, droplets can be sized to contain a cell and at the same time minimize the extracellular volume, resulting in high extracellular concentrations of protein and hence, rapid and sensitive assays. These droplets can be used for enrichment of library elements that can be subjected to secondary mining to optimize a broad range of protein characteristics. Ultra-high throughput screening combines the use of prescreening techniques and computational search methods to maximize library analysis, resulting in the exploration of a very large protein sequence-space.
The manufacture and use of microfluidic devices is well-known as is their use to screen chemical libraries, e.g., comprising polynucleotides, which after microfluidic screening and/or sorting are transformed into a host cell for expression.
In routine Bacillus subtilis transformation procedures, only about 1 in 10,000 cells are actually transformed. Generally, an antibiotic resistance gene is provided together with a gene of interest which prevents untransformed cells from growing in the presence of an added antibiotic. Even though untransformed cells are not able to grow or divide under antibiotic stress, they will usually survive being exposed to the commonly used antibiotics and resume growth once the antibiotic is removed. In fact, many commonly used antibiotics are not really true antibiotics in the sense that they do not kill the cells; most so-called antibiotics would be termed more accurately bacteriostatics.
Due to the relatively low transformation efficiency, a gene library has to be 50,000-fold overscreened to compensate for the total variant dilution. This adds on top of the redundancy of a diversity library, that in itself has to be 5-fold overscreened to cover the diversity.
In theory, nano- to pico-size droplet microfluidics technology allows the screening of as much as 1000 cells per second. However, in real applications this number is reduced by the fact that the cells are diluted during droplet packaging, so that only about 1 in 5 droplet contains a cell. This is done to increase the likelihood, that the droplets contain a maximum of 1 cell when inoculated. A consequence is that an unmanageably large number of cells have to screened in order to cover an entire gene library.
A way to increase the rate of transformed vs. untransformed cells in transformations of microorganisms would be to use a selection principle, whereby the untransformed cells are actually killed, preferably without the need to add anything to the growth medium. Such an improvement of transformation efficiency would be of particular relevance in microfluidic applications.